Recombinant Newcastle disease virus RNA expression systems and vaccines

ABSTRACT

This invention relates to genetically engineered Newcastle disease viruses and viral vectors which express heterologous genes or mutated Newcastle disease viral genes or a combination of viral genes derived from different strains of Newcastle disease virus. The invention relates to the construction and use of recombinant negative strand NDV viral RNA templates which may be used with viral RNA-directed RNA polymerase to express heterologous gene products in appropriate host cells and/or to rescue the heterologous gene in virus particles. In a specific embodiment of the invention, the heterologous gene product is a peptide or protein derived from the genome of a human immunodeficiency virus. The RNA templates of the present invention may be prepared by transcription of appropriate DNA sequences using any DNA-directed RNA polymerase such as bacteriophage T 7 , T 3 , SP 6  polymerase, or eukaryotic polymerase I.

This application is a continuation of application no. 09/576,567, filedMay 22, 2000, now U.S. Pat. No. 6,451,323, which is acontinuation-in-part of application no. 09/152,845, filed Sep. 14, 1998,now U.S. Pat. No. 6,146,642. This application also claims priority fromPCT/US99/21081, filed Sep. 14, 1999. The aforementioned priorityapplications are incorporated by reference herein in their entirety.

1. INTRODUCTION

The invention was made with government support under grant numbers97308MI and 73054MI awarded by the National Institutes of Health. TheGovernment has certain rights in these inventions.

The present invention relates to recombinant Newcastle disease virus RNAtemplates which may be used to express heterologous gene products inappropriate host cell systems and/or to construct recombinant virusesthat express, package, and/or present the heterologous gene product. Theexpression products and chimenic viruses may advantageously be used invaccine formulations. The present invention also relates to geneticallyengineered recombinant Newcastle disease viruses which containmodifications and/or mutations that make the recombinant virus suitablefor use in vaccine formulations, such as an attenuated phenotype orenhanced immunogenicity.

The present invention relates to recombinant Newcastle disease viruseswhich induce interferon and related pathways. The present inventionrelates to the use of the recombinant Newcastle disease viruses andviral vectors against a broad range of pathogens and/or antigens,including tumor specific antigens. The invention is demonstrated by wayof examples in which recombinant Newcastle disease virus RNA templatescontaining heterologous gene coding sequences in the negative-polaritywere constructed. The invention further relates to the construction ofrecombinant Newcastle disease virus RNA templates containingheterologous gene coding sequences in the positive-polarity. Suchheterologous gene sequences include sequences derived from a humanimmunodeficiency virus (HIV).

2. BACKGROUND OF THE INVENTION

A number of DNA viruses have been genetically engineered to direct theexpression of heterologous proteins in host cell systems (e.g., vacciniavirus, baculovirus, etc.). Recently, similar advances have been madewith positive-strand RNA viruses (e.g., poliovirus). The expressionproducts of these constructs, i e., the heterologous gene product or thechimeric virus which expresses the heterologous gene product, arethought to be potentially useful in vaccine formulations (either subunitor whole virus vaccines). One drawback to the use of viruses such asvaccinia for constructing recombinant or chimeric viruses for use invaccines is the lack of variation in its major epitopes. This lack ofvariability in the viral strains places strict limitations on therepeated use of chimeric vaccinia, in that multiple vaccinations willgenerate host-resistance to the strain so that the inoculated viruscannot infect the host. Inoculation of a resistant individual withchimeric vaccinia will, therefore, not induce immune stimulation.

By contrast, the negative-strand RNA viruse, would be attractivecandidates for constructing chimeric viruses for use in vaccines. Thenegative-strand RNA virus, influenza, for example is desirable becauseits wide genetic variability allows for the construction of a vastrepertoire of vaccine formulations which stimulate immunity without riskof developing a tolerance. Recently, construction of infectiousrecombinant or chimeric negative-strand RNA particles was achieved withthe influenza virus (U.S. Pat. No. 5,166,057 to Palese et al.,incorporated herein by reference in its entirety).

2.1. The Newcastle Disease Virus

Virus families containing enveloped single-stranded RNA of thenegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae) or those having segmentedgenomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). TheParamyxoviridae family, described in detail below, and used in theexamples herein, contain the viruses of Newcastle disease virus (NDV),parainfluenza virus, Sendai virus, simian virus 5, and mumps virus.

The Newcastle disease Virus is an enveloped virus containing a linear,single-strand, nonsegmented, negative sense RNA genome. The genomic RNAcontains genes in the order of 3′-NP-P-M-F-HN-L, described in furtherdetail below. The genomic RNA also contains a leader sequence at the 3′end.

The structural elements of the virion include the virus envelope whichis a lipid bilayer derived from the cell plasma membrane. Theglycoprotein, hemagglutinin-neuraminidase (HN), protrudes from theenvelope allowing the virus to contain both hemagglutinin andneuraminidase activities. The fusion glycoprotein (F), which alsointeracts with the viral membrane, is first produced as an inactiveprecursor, then cleaved post-translationally to produce two disulfidelinked polypeptides. The active F protein is involved in penetration ofNDV into host cells by facilitating fusion of the viral envelope withthe host cell plasma membrane. The matrix protein (M), is involved withviral assembly, and interacts with both the viral membrane as well asthe nucleocapsid proteins.

The main protein subunit of the nucleocapsid is the nucleocapsid protein(NP) which confers helical symmetry on the capsid. In association withthe nucleocapsid are the P and L proteins. The phosphoprotein (P), whichis subject to phosphorylation, is thought to play a regulatory role intranscription, and may also be involved in methylation, phosphorylationand polyadenylation. The L gene, which encodes an RNA-dependent RNApolymerase, is required for viral RNA synthesis together with the Pprotein. The L protein, which takes up nearly half of the codingcapacity of the viral genome is the largest of the viral proteins, andplays an important role in both transcription and replication.

The replication of all negative-strand RNA viruses, including NDV, iscomplicated by the absence of cellular machinery required to replicateRNA. Additionally, the negative-strand genome can not be translateddirectly into protein, but must first be transcribed into apositive-strand (mRNA) copy. Therefore, upon entry into a host cell, thegenomic RNA alone cannot synthesize the required RNA-dependent RNApolymerase. The L, P and NP proteins must enter the cell along with thegenome on infection.

It is hypothesized that most or all of the viral proteins thattranscribe NDV mRNA also carry out their replication. The mechanism thatregulates the alternative uses (i.e., transcription or replication) ofthe same complement of proteins has not been clearly identified butappears to involve the abundance of free forms of one or more of thenucleocapsid proteins, in particular, the NP. Directly followingpenetration of the virus, transcription is initiated by the L proteinusing the negative-sense RNA in the nucleocapsid as a template. ViralRNA synthesis is regulated such that it produces monocistronic mRNAsduring transcription.

Following transcription, virus genome replication is the secondessential event in infection by negative-strand RNA viruses. As withother negative-strand RNA viruses, virus genome replication in Newcastledisease virus (NDV) is mediated by virus-specified proteins. The firstproducts of replicative RNA synthesis are complementary copies (i.e.,plus-polarity) of NDV genome RNA (cRNA). These plus-stranded copies(anti-genomes) differ from the plus-strand mRNA transcripts in thestructure of their termini. Unlike the mRNA transcripts, theanti-genomic cRNAs are not capped and methylated at the 5′ termini, andare not truncated and polyadenylated at the 3′ termini. The cRNAs arecoterminal with their negative strand templates and contain all thegenetic information in each genomic RNA segment in the complementaryform. The cRNAs serve as templates for the synthesis of NDVnegative-strand viral genomes (vRNAs).

Both the NDV negative strand genomes (vRNAs) and antigenomes (cRNAs) areencapsidated by nucleocapsid proteins; the only unencapsidated RNAspecies are virus mRNAs. For NDV, the cytoplasm is the site of virus RNAreplication, just as it is the site for transcription. Assembly of theviral components appears to take place at the host cell plasma membraneand mature virus is released by budding.

2.2. Engineering Negative Strand RNA Viruses

The RNA-directed RNA polymerases of animal viruses have been extensivelystudied with regard to many aspects of protein structure and reactionconditions. However, the elements of the template RNA which promoteoptimal expression by the polymerase could only be studied by inferenceusing existing viral RNA sequences. This promoter analysis is ofinterest since it is unknown how a viral polymerase recognizes specificviral RNAs from among the many host-encoded RNAs found in an infectedcell.

Animal viruses containing plus-sense genome RNA can be replicated whenplasmid-derived RNA is introduced into cells by transfection (forexample, Racaniello et al., 1981, Science 214:916-919; Levis, et al.,1986, Cell 44: 137-145). In the case of poliovirus, the purifiedpolymerase will replicate a genome RNA in in vitro reactions and whenthis plus-sense RNA preparation is transfected into cells it isinfectious (Kaplan, et al., 1985, Proc. Natl. Acad. Sci. USA82:8424-8428). However, the template elements which serve astranscription promoter for the poliovirus-encoded polymerase are unknownsince even RNA homopolymers can be copied (Ward, et al., 1988, J. Virol.62: 558-562). SP6 transcripts have also been used to produce modeldefective interfering (DI) RNAs for the Sindbis viral genome. When theRNA is introduced into infected cells, it is replicated and packaged.The RNA sequences which were responsible for both recognition by theSindbis viral polymerase and packaging of the genome into virusparticles were shown to be within 162 nucleotides (nt) of the 5′terminus and 19 nt of the 3′ terminus of the genome (Levis, et al.,1986, Cell 44: 137-145). In the case of brome mosaic virus (BMV), apositive strand RNA plant virus, SP6 transcripts have been used toidentify the promoter as a 134 nt tRNA-like 3′ terminus (Dreher, andHall, 1988, J. Mol. Biol. 201: 31-40). Polymerase recognition andsynthesis were shown to be dependent on both sequence and secondarystructural features (Dreher, et al., 1984, Nature 311: 171-175).

The negative-sense RNA viruses have been refractory to study of thesequence requirements of the replicase. The purified polymerase ofvesicular stomatitis virus is only active in transcription whenvirus-derived ribonucleoprotein complexes (RNPs) are included astemplate (De and Banerjee, 1985, Biochem. Biophys. Res. Commun. 126:40-49; Emerson and Yu, 1975, J. Virol. 15: 1348-1356; Naito andIshihama, 1976, J. Biol. Chem. 251: 4307-4314). With regard to influenzaviruses, it was reported that naked RNA purified from virus was used toreconstitute RNPs. The viral nucleocapsid and polymerase proteins weregel-purified and renatured on the viral RNA using thioredoxin (Szewczyk,et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 7907-7911). However, theseauthors did not show that the activity of the preparation was specificfor influenza viral RNA, nor did they analyze the signals which promotetranscription.

Only recently has it been possible to recover negative strand RNAviruses using a recombinant reverse genetics approach (U.S. Pat. No.5,166,057 to Palese et al.). Although this method was originally appliedto engineer influenza viral genomes (Luytjes et al. 1989, Cell 59:1107-1113; Enami et al. 1990, Proc. Natl. Acad Sci. USA 92:11563-11567), it has been successfully applied to a wide variety ofsegmented and nonsegmented negative strand RNA viruses, including rabies(Schnell et al. 1994, EMBO J. 13:4195-4203); respiratory syncytial virus(Collins et al. 1991, Proc. Natl. Acad. Sci. USA 88:9663-9667); andSendai virus (Park et al. 1991, Proc. Natl. Acad. Sci. USA 88:5537-5541;Kato et al., 1996, Genes Cells 1:569-579). However, this approach hasyet to be applied to Newcastle disease virus RNA genomes.

3. SUMMARY OF THE INVENTION

Recombinant Newcastle disease viral RNA templates are described whichmay be used with RNA-directed RNA polymerase to express heterologousgene products in appropriate host cells and/or to rescue theheterologous gene in virus particles. In one embodiment, the inventionrelates to recombinant Newcastle disease viruses which induce interferonand related pathways. The present invention relates to recombinantNewcastle disease viruses which contain modifications which result inphenotypes which make the recombinant virus more suitable for use invaccine formulations, e.g., attenuated phenotypes and enhancedimmunogenicity. In another embodiment, the present invention relates toengineering recombinant Newcastle disease viruses and viral vectorswhich contain heterologous genes including genes of other viruses,pathogens, cellular genes, tumor antigens etc.

In another embodiment, the present invention relates to engineeringrecombinant Newcastle disease viruses and viral vectors for the use asvaccines. The present invention relates to vaccine formulations suitablefor administration to humans, as well as veterinary uses. The vaccinesof the present invention may be designed for administration to domesticanimals, including cats and dogs; wild animals, including foxes andraccoons; livestock and fowl, including horses, cattle, sheep, turkeysand chickens.

In yet another embodiment, the invention relates to recombinantNewcastle disease viral vectors and viruses which are engineered toencode mutant Newcastle disease viral genes or to encode combinations ofgenes from different strains of Newcastle disease virus. The RNAtemplates of the present are prepared by transcription of appropriateDNA sequences with a DNA-directed RNA polymerase. The resulting RNAtemplates are of the negative-polarity and contain appropriate terminalsequences which enable the viral RNA-synthesizing apparatus to recognizethe template. Alternatively, positive-polarity RNA templates whichcontain appropriate terminal sequences which enable the viralRNA-synthesizing apparatus to recognize the template, may also be used.Expression from positive polarity RNA templates may be achieved bytransfection of plasmids having promoters which are recognized by theDNA-dependent RNA polymerase. For example, plasmid DNA encoding positiveRNA templates under the control of a T7 promoter can be used incombination with the vaccinia virus T7 system.

Bicistronic mRNAs can be constructed to permit internal initiation oftranslation of viral sequences and allow for the expression of foreignprotein coding sequences from the regular terminal initiation site, orvice versa. Alternatively, a foreign protein may be expressed frominternal transcriptional unit in which the transcriptional unit has aninitiation site and polyadenylation site. In another embodiment, theforeign gene is inserted into an NDV gene such that the resultingexpressed protein is a fusion protein.

The recombinant mutant Newcastle disease viral RNA templates of thepresent invention may be used to transfect transformed cell lines thatexpress the RNA dependent RNA-polymerase and allow for complementation.Alternatively, a plasmid expressing from an appropriate promoter, can beused for virus specific (chimeric) RNA transfection. Complementation mayalso be achieved with the use of a helper virus which provides the RNAdependent RNA-polymerase. Additionally, a non-virus dependentreplication system for Newcastle disease virus is also described. Theminimum subset of Newcastle disease virus proteins needed for specificreplication and expression of the virus are the three proteins, L, P andNP, which can be expressed from plasmids by a vaccinia virus T7 system.In yet another embodiment, when plasmids encoding the antigenomic copyof the NDV genome are used to supply the viral genome, the minimumsubset of Newcastle disease virus proteins needed for specificreplication and expression of the virus are the L and P proteins. Whenthe antigenomic copy of the NDV genome is transcribed, th NP polymeraseprotein is the first protein transcribed, thus it is not necessary toadditionally provide the NP polymerase in trans.

The expression products and/or chimeric virions obtained mayadvantageously be utilized in vaccine formulations. The expressionproducts and chimeric virions of the present invention may be engineeredto create vaccines against a broad range of pathogens, including viralantigens, tumor antigens and auto antigens involved in autoimmunedisorders. In particular, the chimeric virions of the present inventionmay be engineered to create anti-HIV vaccines, wherein an immunogenicpolypeptide from gp160, and/or from internal proteins of HIV isengineered into the glycoprotein HN protein to construct a vaccine thatis able to elicit both vertebrate humoral and cell-mediated immuneresponses. The use of recombinant Newcastle disease virus for thispurpose is especially attractive since Newcastle disease virus is notpathogenic in humans. The use of recombinant Newcastle disease virus fordelivering tumor antigens is particularly attractive given the knownantineoplastic, and immunopotentiating properties of the virus.

3.1. Definitions

As used herein, the following terms will have the meanings indicated:

-   -   cRNA=anti-genomic RNA    -   HIV=human immunodefiency virus    -   L=large protein    -   M=matrix protein (lines inside of envelope)    -   MDCK=Madin Darby canine kidney cells    -   MDBK=Madin Darby bovine kidney cells    -   moi=multiplicity of infection    -   NA=neuraminidase (envelope glycoprotein)    -   NDV=Newcastle disease Virus    -   NP=nucleoprotein (associated with RNA and required for        polymerase activity)    -   NS=nonstructural protein (function unknown)    -   nt=nucleotide    -   PA, PB1, PB2=RNA-directed RNA polymerase components    -   RNP=ribonucleoprotein    -   rRNP=recombinant RNP    -   vRNA=genomic virus RNA    -   WSN=influenza A/WSN/33 virus    -   WSN-HK virus: reassortment virus containing seven genes from WSN        virus and the NA gene from influenza A/HK/8/68 virus

4. DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the NDV minigenome. Top illustrationdepicts the PNDVCAT plasmid including the T7 promoter; the 5′ terminalsequence (5′ end of genomic RNA, 191nt); the inserted nucleotides(CTTAA); 667nt of CAT ORF; the 3′ terminal sequence (3′ end of genomicRNA, 121 nt) the Bbs1 and nuclease sites. Lower illustration depicts thechimeric NDV-CAT RNA resulting from in vitro transcription. As a resultof the NDV-based amplification and transcription of the NDV-CAT chimericminigenome, CAT activity is detected in the transfected cells.

FIGS. 2A-C. Schematic representation of the PTMI expression vectors.

PTM1-NP encodes the NDV NP protein.

PTM1-P encodes the NDV P protein.

PTM1-L encodes the NDV L protein.

FIG. 3. RNA sequence of NDV 5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2)non-coding terminal regions (plus-sense). Sequences 5′ to the CAT generepresent 121lnt of the 5′ non-coding terminal region of NDV plus sensegenome comprising 65nt of the leader sequence (in bold) followed by 56ntof the NP gene UTR. Sequences 3′ to the CAT gene represent insertednucleotides cuuaa (in lower case) and 191nt of the non-coding terminalregion of NDV plus sense genome comprising 127nt of the UTR of the Lgene followed by 64nt of the trailer region (in bold).

FIG. 4A-B Schematic representation of a structure of recombinant NDVclones. FIG. 4B, representation of infectious NDV expressing HIV Env andGag. Top panel, HIV Env And Gag are between the M and L genes. Lowerpanel, HIV Env and Gag are 3′ to the NP Gene.

FIG. 5 Schematic representation of the 3′ and 5′ termini of NDV asaligned with sequence of Kurilla et al. 1985 Virology 145:203-212 (3′termini) and Yusoffet al. 1987 Nucleic Acids Research 15:3961-3976 (5′termini) (top: SEQ ID NO: 3 aligned with SEQ ID NO: 4 and bottom: SEQ IDNO: 5 aligned with SEQ ID NO: 6).

FIG. 6 Plasmid-based reverse genetics method for NDV-based expression ofa foreign gene. Cells are infected with a recombinant vaccinia virusexpressing T7 polymerase. In addition, cells are transfected with 1)plasmid DNAs encoding the L, NP and P proteins of NDV under thetranscriptional control of a T7 promoter (pTM1-L, pTM1-NP and pTM1-P,respectively) and 2) a plasmid DNA encoding a chimeric NDV-CATminigenome under the transcriptional control of a T7 promoter(pT7-NDV-CAT-RB). The proper 3′ end of the NDV-CAT minigenome isachieved by relying on the cleavage facilitated via a ribozyme sequence(RB). Amplification and transcription of the NDV-CAT chimeric minigenomeresults in CAT activity detectable in the transfected cells. Thenoncoding regions at the 3′ and 5′ ends of the NDV-CAT minigenome arerepresented as black boxes.

FIG. 7 Rescue of NDV from synthetic DNA. Cells are infected with arecombinant vaccinia virus expressing T7 polymerase. In addition, cellsare transfected with 1) plasmid DNAs encoding the L, NP and P proteinsof NDV under the transcriptional control of a T7 promoter (pTM1-L,pTM1-NP and pTM1-P, respectively) and 2) a plasmid DNA encoding the NDVantigenome under the transcriptional control of a T7 promoter(pT7-NDV+-RB). The proper 3′ end of the NDV antigenome is achieved byrelying on the cleavage facilitated via a ribozyme sequence (RB).Amplification and transcription of the NDV antigenome results in therescue of infectious NDV viruses. The noncoding regions at the 3′ and 5′ends of the NDV antigenome are represented as black boxes.

FIG. 8 NDV-based expression of a foreign gene inserted as an internaltranscriptional unit into the NDV antigenome. Cells are infected with arecombinant vaccinia virus expressing T7 polymerase. In addition, cellsare transfected with 1) plasmid DNAs encoding the L, NP and P proteinsof NDV under the transcriptional control of a T7 promoter (pTM1-L,pTM1-NP and pTM1-P, respectively) and 2) a plasmid DNA encoding achimeric NDV-CAT antigenome under the transcriptional control of a T7promoter (pT7-NDV-CAT-RB). In the chimeric NDV-CAT antigenome, the CATopen reading frame substitutes the naturally occurring HN open readingframe of the wild-type NDV antigenome. The proper 3′ end of the chimericNDV-CAT antigenome is achieved by relying on the cleavage facilitatedvia a ribozyme sequence (RB). Amplification and transcription of thechimeric NDV-CAT antigenome results in CAT activity detectable in thetransfected cells. The noncoding regions at the 3′ and 5′ ends of thechimeric NDV-CAT antigenome are represented as black boxes.

5. DESCRIPTION OF THE INVENTION

This invention relates to genetically engineered Newcastle diseaseviruses and viral vectors which express heterologous genes or mutatedNewcastle disease viral genes or a combination of viral genes derivedfrom different strains of Newcastle disease virus. The invention relatesto the construction and use of recombinant negative strand NDV viral RNAtemplates which may be used with viral RNA-directed RNA polymerase toexpress heterologous gene products in appropriate host cells and/or torescue the heterologous gene in virus particles. In a specificembodiment of the invention, the heterologous gene product is a peptideor protein derived from the genome of a human immunodeficiency virus.The RNA templates of the present invention may be prepared either invitro or in vivo by transcription of appropriate DNA sequences using aDNA-directed RNA polymerase such as bacteriophage T7, T3, the SP6polymerase or a eukaryotic polymerase such as polymerase I.

The recombinant RNA templates may be used to transfectcontinuous/transfected cell lines that express the RNA-directed RNApolymerase proteins allowing for complementation, as demonstrated by wayof working examples in which RNA transcripts of cloned DNA containingthe coding region—in negative sense orientation—of the chloramphenicolacetyltransferase (CAT) gene, flanked by the 5′ terminal and the 3′terminal nucleotides of the NDV-CL (California strain/11914/1944-likestrain) (Meindl et al., 1974 Virology 58: 457-463) RNA were transfectedinto cells expressing the NDV polymerase proteins. In a preferredembodiment, a non-virus dependent replication system is used to recoverchimeric NDV, in which plasmid DNA encoding the NDV genome or antigenomeis coexpressed with plasmid DNA encoding the minimum subset of Newcastledisease virus proteins needed for specific replication and expression ofthe virus, as demonstrated by way of working example as described infra.

The ability to reconstitute NDV in vivo allows the design of novelchimeric NDV viruses which express foreign genes or which express mutantNDV genes. The ability to reconstitute NDV in vivo also allows thedesign of novel chimeric NDVs which express genes from different strainsof NDV. One way to achieve this goal involves modifying existing NDVgenes. For example, the HN gene may be modified to contain foreignsequences in its external domains. Where the heterologous sequence areepitopes or antigens of pathogens, these chimeric viruses may be used toinduce a protective immune response against the disease agent from whichthese determinants are derived.

In accordance with the present invention, a chimeric RNA is constructedin which a coding sequence derived from the gp160 coding region of humanimmunodeficiency virus is inserted into the HN coding sequence of NDV,and chimeric virus produced from transfection of this chimeric RNAsegment into a host cell infected with wild-type NDV. Further, such achimeric virus should be capable of eliciting both a vertebrate humoraland cell-mediated immune response. The present invention further relatesto the induction of interferon and related pathways by recombinant orchimeric NDV viruses.

The present invention relates to the use of viral vectors and chimericviruses of the invention to formulate vaccines against a broad range ofviruses and/or antigens including tumor antigens. The viral vectors andchimeric viruses of the present invention may be used to modulate asubject's immune system by stimulating a humoral immune response, acellular immune response or by stimulating tolerance to an antigen. Asused herein, a subject means: humans, primates, horses, cows, sheep,pigs, goats, dogs, cats, avian species and rodents. When delivering,tumor antigens, the invention may be used to treat subjects havingdisease amenable to immunity mediated rejection, such as non-solidtumors or solid tumors of small size. It is also contemplated thatdelivery of tumor antigens by the viral vectors and chimeric virusesdescribed herein will be useful for treatment subsequent to removal oflarge solid tumors. The invention may also be used to treat subjects whoare suspected of having cancer.

The invention may be divided into the following stages solely for thepurpose of description and not by way of limitation: (a) construction ofrecombinant RNA templates; (b) expression of heterologous gene productsusing the recombinant RNA templates; and (c) rescue of the heterologousgene in recombinant virus particles. For clarity of discussion, theinvention is described in the working Examples using NDV-CL (Californiastrain/11914/1944-like strain), however any strain of NDV may beutilized.

5.1. Construction of the Recombinant RNA Templates

A specific embodiment of the present invention is the Applicants'identification of the correct nucleotide sequence of the 5′ and 3′termini of the negative-sense genomes RNA of NDV. The nucleotidesequence of the 5′ and 3′ termini of the NDV negative-sense genome RNAof the present invention differs significantly from the NDV 3′ terminisequence previously disclosed as shown in FIG. 5. The identification ofthe correct nucleotide sequence of the NDV 5′ and 3′ termini allows forthe first time the engineering of recombinant NDV RNA templates, theexpression of the recombinant RNA templates and the rescue ofrecombinant NDV particles. The present invention encompasses not only 5′and 3′ termini having the nucleotide sequence as shown in FIG. 5, butalso encompasses any modifications or mutations to the termini or anyfragments thereof that still retain the function of the wildtype terminii.e., the signals required for the viral RNA-synthesizing apparatus torecognize the template.

Heterologous gene coding sequences flanked by the complement of theviral polymerase binding site/promoter, e.g., the complement of 3′-NDVvirus terminus of the present invention, or the complements of both the3′- and 5′-NDV virus termini may be constructed using techniques knownin the art. The resulting RNA templates may be of the negative-polarityand contain appropriate terminal sequences which enable the viralRNA-synthesizing apparatus to recognize the template. Alternatively,positive-polarity RNA templates which contain appropriate terminalsequences which enable the viral RNA-synthesizing apparatus to recognizethe template, may also be used. Recombinant DNA molecules containingthese hybrid sequences can be cloned and transcribed by a DNA-directedRNA polymerase, such as bacteriophage T7, T3, the SP6 polymerase oreukaryotic polymerase such as polymerase I and the like, to produce invitro or in vivo the recombinant RNA templates which possess theappropriate viral sequences that allow for viral polymerase recognitionand activity.

In yet another embodiment, virtually any heterologous sequence may beconstructed into the chimeric viruses of the present invention,including but not limited to antigens, such as 1) antigens that arecharacteristic of a pathogen; 2) antigens that are characteristic ofautoimmune disease; 3) antigens that are characteristic of an allergen;and 4) antigens that are characteristic of a tumor. For example,heterologous gene sequences that can be engineered into the chimericviruses of the invention include, but are not limited to, epitopes ofhuman immunodeficiency virus (HIV) such as gp160; hepatitis B virussurface antigen (HBsAg); the glycoproteins of herpes virus (e.g., gD,gE); VP1 of poliovirus; and antigenic determinants of nonviral pathogenssuch as bacteria and parasites to name but a few.

Antigens that are characteristic of autoimmune disease typically will bederived from the cell surface, cytoplasm, nucleus, mitochondria and thelike of mammalian tissues, including antigens characteristic of diabetesmellitus, multiple sclerosis, systemic lupus erythematosus, rheumatoidarthritis, pernicious anemia, Addison's disease, scleroderma, autoimmuneatrophic gastritis, juvenile diabetes, and discoid lupus erythromatosus.

Antigens that are allergens are generally proteins or glycoproteins,including antigens derived from pollens, dust, molds, spores, dander,insects and foods.

Antigens that are characteristic of tumor antigens typically will bederived from the cell surface, cytoplasm, nucleus, organelles and thelike of cells of tumor tissue. Examples include antigens characteristicof tumor proteins, including proteins encoded by mutated oncogenes;viral proteins associated with tumors; and glycoproteins. Tumorsinclude, but are not limited to, those derived from the types of cancer:lip, nasopharynx, pharynx and oral cavity, esophagus, stomach, colon,rectum, liver, gall bladder, pancreas, larynx, lung and bronchus,melanoma of skin, breast, cervix, uterine, ovary, bladder, kidney,uterus, brain and other parts of the nervous system, thyroid, prostate,testes, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma andleukemia.

In one specific embodiment of the invention, the heterologous sequencesare derived from the genome of human immunodeficiency virus (HIV),preferably human immunodeficiency virus-1 or human immunodeficiencyvirus-2. In another embodiment of the invention, the heterologous codingsequences may be inserted within an NDV gene coding sequence such that achimeric gene product is expressed which contains the heterologouspeptide sequence within the NDV viral protein. In such an embodiment ofthe invention, the heterologous sequences may also be derived from thegenome of a human immunodeficiency virus, preferably of humanimmunodeficiency virus-1 or human immunodeficiency virus-2.

In instances whereby the heterologous sequences are HIV-derived, suchsequences may include, but are not limited to sequences derived from theenv gene (i.e., sequences encoding all or part of gp160, gp120, and/orgp41), the pol gene (i.e., sequences encoding all or part of reversetranscriptase, endonuclease, protease, and/or integrase), the gag gene(i.e., sequences encoding all or part of p7, p6, p55, p17/18, p24/25)tat, rev, nef, vif, vpu, vpr, and/or vpx.

In yet another embodiment, heterologous gene sequences that can beengineered into the chimeric viruses include those that encode proteinswith immunopotentiating activities. Examples of immunopotentiatingproteins include, but are not limited to, cytokines, interferon type 1,gamma interferon, colony stimulating factors, interleukin-1, -2, -4, -5,-6, -12.

One approach for constructing these hybrid molecules is to insert theheterologous coding sequence into a DNA complement of an NDV gene sothat the heterologous sequence is flanked by the viral sequencesrequired for viral polymerase activity; i e., the viral polymerasebinding site/promoter, hereinafter referred to as the viral polymerasebinding site, and a polyadenylation site. In a preferred embodiment, theheterologous coding sequence is flanked by the viral sequences thatcomprise the replication promoters of the 5′ and 3′ termini, the genestart and gene end sequences, and the packaging signals that are foundin the 5′ and/or the 3′ termini. In an alternative approach,oligonucleotides encoding the viral polymerase binding site, e.g., thecomplement of the 3′-terminus or both termini of the virus genomicsegments can be ligated to the heterologous coding sequence to constructthe hybrid molecule. The placement of a foreign gene or segment of aforeign gene within a target sequence was formerly dictated by thepresence of appropriate restriction enzyme sites within the targetsequence. However, recent advances in molecular biology have lessenedthis problem greatly. Restriction enzyme sites can readily be placedanywhere within a target sequence through the use of site-directedmutagenesis (e.g., see, for example, the techniques described by Kunkel,1985, Proc. Natl. Acad. Sci. U.S.A. 82;488). Variations in polymerasechain reaction (PCR) technology, described infra, also allow for thespecific insertion of sequences (i.e., restriction enzyme sites) andallow for the facile construction of hybrid molecules. Alternatively,PCR reactions could be used to prepare recombinant templates without theneed of cloning. For example, PCR reactions could be used to preparedouble-stranded DNA molecules containing a DNA-directed RNA polymerasepromoter (e.g., bacteriophase T3, T7 or SP6) and the hybrid sequencecontaining the heterologous gene and the NDV polymerase binding site.RNA templates could then be transcribed directly from this recombinantDNA. In yet another embodiment, the recombinant RNA templates may beprepared by ligating RNAs specifying the negative polarity of theheterologous gene and the viral polymerase binding site using an RNAligase. Sequence requirements for viral polymerase activity andconstructs which may be used in accordance with the invention aredescribed in the subsections below.

5.1.1. Insertion of the Heterologous Gene Sequence Into the HN, P, NP,M, F, L Genes

The gene segments coding for the HN, P, NP, M, F, or L proteins may beused for the insertion of heterologous gene products. Insertion of aforeign gene sequence into any of these segments could be accomplishedby either a complete replacement of the viral coding region with theforeign gene or by a partial replacement. Complete replacement wouldprobably best be accomplished through the use of PCR-directedmutagenesis. Briefly, PCR-primer A would contain, from the 5′ to 3′end:a unique restriction enzyme site, such as a class IIS restriction enzymesite (i.e., a “shifter” enzyme; that recognizes a specific sequence butcleaves the DNA either upstream or downstream of that sequence); astretch of nucleotides complementary to a region of the NDV gene; and astretch of nucleotides complementary to the carboxy-terminus codingportion of the foreign gene product. PCR-primer B would contain from the5′ to 3′ end: a unique restriction enzyme site; a stretch of nucleotidescomplementary to a NDV gene; and a stretch of nucleotides correspondingto the 5′ coding portion of the foreign gene. After a PCR reaction usingthese primers with a cloned copy of the foreign gene, the product may beexcised and cloned using the unique restriction sites. Digestion withthe class IIS enzyme and transcription with the purified phagepolymerase would generate an RNA molecule containing the exactuntranslated ends of the NDV gene with a foreign gene insertion. In analternate embodiment, PCR-primed reactions could be used to preparedouble-stranded DNA containing the bacteriophage promoter sequence, andthe hybrid gene sequence so that RNA templates can be transcribeddirectly without cloning.

5.1.2. Insertion of the Heterologous Gene Sequence into the HN Gene

The hemagglutinin and neuraminidase activities of NDV are coded for by asingle gene, HN. The HN protein is a major surface glycoprotein of thevirus. For a variety of viruses, such as influenza, the hemagglutininand neuraminidase proteins have been demonstrated to contain a number ofantigenic sites. Consequently, this protein is a potential target forthe humoral immune response after infection. Therefore, substitution ofantigenic sites within HN with a portion of a foreign protein mayprovide for a vigorous humoral response against this foreign peptide. Ifa sequence is inserted within the HN molecule and it is expressed on theoutside surface of the HN it will be immunogenic. For example, a peptidederived from gp160 of HIV could replace an antigenic site of the HNprotein, resulting in the elicitation of both a humoral immune response.In a different approach, the foreign peptide sequence may be insertedwithin the antigenic site without deleting any viral sequences.Expression products of such constructs may be useful in vaccines againstthe foreign antigen, and may indeed circumvent a problem discussedearlier, that of propagation of the recombinant virus in the vaccinatedhost. An intact HN molecule with a substitution only in antigenic sitesmay allow for HN function and thus allow for the construction of aviable virus. Therefore, this virus can be grown without the need foradditional helper functions. The virus may also be attenuated in otherways to avoid any danger of accidental escape.

Other hybrid constructions may be made to express proteins on the cellsurface or enable them to be released from the cell. As a surfaceglycoprotein, the HN has an amino-terminal cleavable signal sequencenecessary for transport to the cell surface, and a carboxy-terminalsequence necessary for membrane anchoring. In order to express an intactforeign protein on the cell surface it may be necessary to use these HNsignals to create a hybrid protein. In this case, the fusion protein maybe expressed as a separate fusion protein from an additional internalpromoter. Alternatively, if only the transport signals are present andthe membrane anchoring domain is absent, the protein may be secreted outof the cell.

5.1.3. Construction of Bicistronic RNA and Heterologous ProteinExpression

Bicistronic mRNA could be constructed to permit internal initiation oftranslation of viral sequences and allow for the expression of foreignprotein coding sequences from the regular terminal initiation site.Alternatively, a bicistronic mRNA sequence may be constructed whereinthe viral sequence is translated from the regular terminal open readingframe, while the foreign sequence is initiated from an internal site.Certain internal ribosome entry site (IRES) sequences may be utilized.The IRES sequences which are chosen should be short enough to notinterfere with Newcastle disease virus packaging limitations. Thus, itis preferable that the IRES chosen for such a bicistronic approach be nomore than 500 nucleotides in length, with less than 250 nucleotidesbeing preferred. Further, it is preferable that the IRES utilized notshare sequence or structural homology with picomaviral elements.Preferred IRES elements include, but are not limited to the mammalianBiP IRES and the hepatitis C virus IRES.

Alternatively, a foreign protein may be expressed from a new internaltranscriptional unit in which the transcriptional unit has an initiationsite and polyadenylation site. In another embodiment, the foreign geneis inserted into an NDV gene such that the resulting expressed proteinis a fusion protein.

5.2. Expression of Heterologous Gene Products Using Recombinant RNATemplate

The recombinant templates prepared as described above can be used in avariety of ways to express the heterologous gene products in appropriatehost cells or to create chimeric viruses that express the heterologousgene products. In one embodiment, the recombinant template can be usedto transfect appropriate host cells, may direct the expression of theheterologous gene product at high levels. Host cell systems whichprovide for high levels of expression include continuous cell lines thatsupply viral functions such as cell lines superinfected with NDV, celllines engineered to complement NDV functions, etc.

In an alternate embodiment of the invention, the recombinant templatesmay be used to transfect cell lines that express a viral polymeraseprotein in order to achieve expression of the heterologous gene product.To this end, transformed cell lines that express a polymerase proteinsuch as the L protein may be utilized as appropriate host cells. Hostcells may be similarly engineered to provide other viral functions oradditional functions such as NP or HN.

In another embodiment, a helper virus may provide the RNA polymeraseprotein utilized by the cells in order to achieve expression of theheterologous gene product.

In yet another embodiment, cells may be transfected with vectorsencoding viral proteins such as the NP, P and L proteins. Examples ofsuch vectors are illustrated in FIGS. 2A-2C.

5.3. Preparation of Chimeric Negative Strand RNA Virus

In order to prepare chimeric virus, modified NDV virus RNAs, cDNAs orRNA coding for the NDV genome and/or foreign proteins in the plus orminus sense may be used to transfect cells which provide viral proteinsand functions required for replication and rescue or are also infectedwith a “parent” NDV virus. In an alternative approach, plasmids encodingthe genomic or antigenomic NDV RNA, either wild type or modified, may beco-transfected into host cells with plasmids encoding viral polymeraseproteins, e.g., NP, P or L. In another embodiment, plasmids encoding theantigenomic NDV RNA may be co-transfected with plasmids encoding viralpolymerase proteins P and L, as the NP polymerase protein is the firstprotein transcribed by the antigenomic copy of the NDV genome, it is notnecessary to additionally provide the NP polymerase in trans.

In an embodiment of the present invention, the reverse geneticstechnique may be utilized to engineer the chimeric negative strand RNAvirus, this technique involves the preparation of synthetic recombinantviral RNAs that contain the non-coding regions of the negative strandvirus RNA which are essential for the recognition by viral polymerasesand for packaging signals necessary to generate a mature virion. Thesynthetic recombinant plasmid DNAs and RNAs can be replicated andrescued into infectious virus particles by any number of techniquesknown in the art, as described in U.S. Pat. No. 5,166,057 issued Nov.24, 1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in EuropeanPatent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. patentapplication Ser. No. 09/152,845; in International Patent PublicationsPCT WO97/12032 published Apr. 3, 1997; WO96/34625 published Nov. 7,1996; in European Patent Publication EP-A780475; WO 99/02657 publishedJan. 21, 1999; WO 98/53078 published Nov. 26, 1998; WO 98/02530published Jan. 22, 1998; WO 99/15672 published Apr. 1, 1999; WO 98/13501published Apr. 2, 1998; WO 97/06270 published Feb. 20, 1997; and EPO 78047SA1 published Jun. 25, 1997, each of which is incorporated byreference herein in its entirety.

There are a number of different approaches which may be used to applythe reverse genetics approach to rescue negative strand RNA viruses.First, the recombinant RNAs are synthesized from a recombinant DNAtemplate and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. In another approach, a more efficient transfectionis achieved if the viral polymerase proteins are present duringtranscription of the synthetic RNAs either in vitro or in vivo. Withthis approach the synthetic RNAs may be transcribed from cDNA plasmidswhich are either co-transcribed in vitro with cDNA plasmids encoding thepolymerase proteins, or transcribed in vivo in the presence ofpolymerase proteins, i.e., in cells which transiently or constitutivelyexpress the polymerase proteins.

In an alternate embodiment, a combination of reverse genetics techniquesand reasserting techniques can be used to engineer attenuated viruseshaving the desired epitopes in segmented RNA viruses. For example, anattenuated virus (generated by natural selection, mutagenesis or byreverse genetics techniques) and a strain carrying the desired vaccineepitope (generated by natural selection, mutagenesis or by reversegenetics techniques) can be co-infected in hosts that permitreassortment of the segmented genomes. Reassortants that display boththe attenuated phenotype and the desired epitope can then be selected.

Following reassortment, the novel viruses may be isolated and theirgenomes identified through hybridization analysis. In additionalapproaches described herein, the production of infectious chimeric virusmay be replicated in host cell systems that express an NDV viralpolymerase protein (e.g., in virus/host cell expression systems;transformed cell lines engineered to express a polymerase protein,etc.), so that infectious chimeric virus are rescued. In this instance,helper virus need not be utilized since this function is provided by theviral polymerase proteins expressed.

In accordance with the present invention, any technique known to thoseof skill in the art may be used to achieve replication and rescue ofchimeric viruses. One approach involves supplying viral proteins andfunctions required for replication in vitro prior to transfecting hostcells. In such an embodiment, viral proteins may be supplied in the formof wildtype virus, helper virus, purified viral proteins orrecombinantly expressed viral proteins. The viral proteins may besupplied prior to, during or post transcription of the synthetic cDNAsor RNAs encoding the chimeric virus. The entire mixture may be used totransfect host cells. In another approach, viral proteins and functionsrequired for replication may be supplied prior to or duringtranscription of the synthetic cDNAs or RNAs encoding the chimericvirus. In such an embodiment, viral proteins and functions required forreplication are supplied in the form of wildtype virus, helper virus,viral extracts, synthetic cDNAs or RNAs which express the viral proteinsare introduced into the host cell via infection or transfection. Thisinfection/transfection takes place prior to or simultaneous to theintroduction of the synthetic cDNAs or RNAs encoding the chimeric virus.

In a particularly desirable approach, cells engineered to express allNDV viral genes may result in the production of infectious chimericvirus which contain the desired genotype; thus eliminating the need fora selection system. Theoretically, one can replace any one of the sixgenes or part of any one of the six genes of NDV with a foreignsequence. However, a necessary part of this equation is the ability topropagate the defective virus (defective because a normal viral geneproduct is missing or altered). A number of possible approaches exist tocircumvent this problem. In one approach a virus having a mutant proteincan be grown in cell lines which are constructed to constitutivelyexpress the wild type version of the same protein. By this way, the cellline complements the mutation in the virus. Similar techniques may beused to construct transformed cell lines that constitutively express anyof the NDV genes. These cell lines which are made to express the viralprotein may be used to complement the defect in the recombinant virusand thereby propagate it. Alternatively, certain natural host rangesystems may be available to propagate recombinant virus.

In yet another embodiment, viral proteins and functions required forreplication may be supplied as genetic material in the form of syntheticcDNAs or RNAs so that they are co-transcribed with the synthetic cDNAsor RNAs encoding the chimeric virus. In a particularly desirableapproach, plasmids which express the chimeric virus and the viralpolymerase and/or other viral functions are co-transfected into hostcells, as described in the Examples, see Section 11 supra.

Another approach to propagating the recombinant virus may involveco-cultivation with wild-type virus. This could be done by simply takingrecombinant virus and co-infecting cells with this and another wild-typevirus (preferably a vaccine strain). The wild-type virus shouldcomplement for the defective virus gene product and allow growth of boththe wild-type and recombinant virus. Alternatively, a helper virus maybe used to support propagation of the recombinant virus.

In another approach, synthetic templates may be replicated in cellsco-infected with recombinant viruses that express the NDV viruspolymerase protein. In fact, this method may be used to rescuerecombinant infectious virus in accordance with the invention. To thisend, the NDV polymerase protein may be expressed in any expressionvector/host cell system, including but not limited to viral expressionvectors (e.g., vaccinia virus, adenovirus, baculovirus, etc.) or celllines that express a polymerase protein (e.g., see Krystal et al., 1986,Proc. Natl. Acad. Sci. USA 83: 2709-2713). Moreover, infection of hostcells expressing all six NDV proteins may result in the production ofinfectious chimeric virus particles. This system would eliminate theneed for a selection system, as all recombinant virus produced would beof the desired genotype. It should be noted that it may be possible toconstruct a recombinant virus without altering virus viability. Thesealtered viruses would then be growth competent and would not need helperfunctions to replicate.

5.4. Vaccine Formulations Using the Chimeric Viruses

The invention encompasses vaccine formulations comprising the engineerednegative strand RNA virus of the present invention. The inventionencompasses the use of recombinant NDV viruses which have been modifiedin vaccine formulations to confer L protection against NDV infection. Inyet another embodiment, the recombinant NDV viruses of the presentinvention may be used as a vehicle to express foreign epitopes thatinduce a protective response to any of a variety of pathogens.

The invention encompasses vaccine formulations to be administered tohumans and animals. In particular, the invention encompasses vaccineformulations to be administered to domestic animals, including dogs andcats; wild animals, including foxes and raccoons; and livestock,including cattle, horses, and pigs, sheep and goats; and fowl, includingchicken and turkey.

The invention encompasses vaccine formulations which are useful againstavian disease causing agents including NDV, Marek's Disease Virus (MDV),Infectious Bursal Disease Virus (IBDV), Infectious Bronchitis Virus(IBV), Infectious Bursitis Virus, Chicken Anemia Virus (CAV), InfectiousLaryngotracheitis Virus (ILV), Avian Leukosis Virus (ALV),Reticuloendotheliosis Virus (RV) and Avian Influenza Virus.

In another embodiment, the invention encompasses vaccine formulationswhich are useful against domestic disease causing agents includingrabies virus, feline leukemia virus (FLV) and canine distemper virus. Inyet another embodiment, the invention encompasses vaccine formulationswhich are useful to protect livestock against vesicular stomatitisvirus, rabies virus, rinderpest virus, swinepox virus, and further, toprotect wild animals against rabies virus.

Attenuated viruses generated by the reverse genetics approach can beused in the vaccine and pharmaceutical formulations described herein.Reverse genetics techniques can also be used to engineer additionalmutations to other viral genes important for vaccine production—i.e.,the epitopes of useful vaccine strain variants can be engineered intothe attenuated virus. Alternatively, completely foreign epitopes,including antigens derived from other viral or non-viral pathogens canbe engineered into the attenuated strain. For example, antigens ofnon-related viruses such as HIV (gp160, gp120, gp41) parasite antigens(e.g., malaria), bacterial or fungal antigens or tumor antigens can beengineered into the attenuated strain. Alternatively, epitopes whichalter the tropism of the virus in vivo can be engineered into thechimeric attenuated viruses of the invention.

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in vaccines. Preferably,epitopes that induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention include, but are not limited to influenzaglycoproteins, in particular, hemagglutinin H5, H7, Marek's DiseaseViral epitopes; epitopes of Infectious Bursal Disease Virus (IBDV),Infectious Bronchitis Virus (IBV), Chicken Anemia Virus (CAV),Infectious Laryngotracheitis Virus (ILV), Avian Leukosis Virus (ALV),Reticuloendotheliosis Virus (RV), Avian Influenza Virus (AIV), rabiesvirus, feline leukemia virus, canine distemper virus, vesicularstomatitis virus, rinderpest virus, and swinepox virus (see Fields etal. (ed.), 1991, Fundamental Virology, Second Edition, Raven Press, NewYork, incorporated by reference herein in its entirety).

In yet another embodiment, heterologous gene sequences that can beengineered into the chimeric viruses include those that encode proteinswith immunopotentiating activities. Examples of immunopotentiatingproteins include, but are not limited to, cytokines, interferon type 1,gamma interferon, colony stimulating factors, interleukin-1, -2, -4, -5,-6, -12.

In addition, heterologous gene sequences that can be constructed intothe chimeric viruses of the invention for use in vaccines include butare not limited to sequences derived from a human immunodeficiency virus(HIV), preferably type 1 or type 2. In a preferred embodiment, animmunogenic HIV-derived peptide which may be the source of an antigenmay be constructed into a chimeric NDV that may then be used to elicit avertebrate immune response. Such HIV-derived peptides may include, butare not limited to sequences derived from the env gene (i.e., sequencesencoding all or part of gp160, gp120, and/or gp41), the pol gene (i.e.,sequences encoding all or part of reverse transcriptase, endonuclease,protease, and/or integrase), the gag gene (i.e., sequences encoding allor part of p7, p6, p55, p17/18, p24/25), tat, rev, nef, vif, vpu, vpr,and/or vpx.

Other heterologous sequences may be derived from hepatitis B virussurface antigen (HBsAg); hepatitis A or C virus surface antigens, theglycoproteins of Epstein Barr virus; the glycoproteins of humanpapillomavirus; the glycoproteins of respiratory syncytial virus,parainfluenza virus, Sendai virus, simianvirus 5 or mumps virus; theglycoproteins of influenza virus; the glycoproteins of herpes virus(e.g. gD, gE); VP1 of poliovirus; antigenic determinants of non-viralpathogens such as bacteria and parasites, to name but a few. In anotherembodiment, all or portions of immunoglobulin genes may be expressed.For example, variable regions of anti-idiotypic immunoglobulins thatmimic such epitopes may be constructed into the chimeric viruses of theinvention.

Other heterologous sequences may be derived from tumor antigens, and theresulting chimeric viruses be used to generate an immune responseagainst the tumor cells leading to tumor regression in vivo. Thesevaccines may be used in combination with other therapeutic regimens,including but not limited to chemotherapy, radiation therapy, surgery,bone marrow transplantation, etc. for the treatment of tumors. Inaccordance with the present invention, recombinant viruses may beengineered to express tumor-associated antigens (TAAs), including butnot limited to, human tumor antigens recognized by T cells (Robbins andKawakami, 1996, Curr. Opin. Immunol. 8:628-636, incorporated herein byreference in its entirety), melanocyte lineage proteins, includinggp100, MART-1/MelanA, TRP-1 (gp75), tyrosinase; Tumor-specific widelyshared antigens, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-1,N-acetylglucosaminyltransferase-V, p15; Tumor-specific mutated antigens,β-catenin, MUM-1, CDK4; Nomelanoma antigens for breast, ovarian,cervical and pancreatic carcinoma, HER-2/neu, human papillomavirus-E6,-E7, MUC-1.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.Additionally, as NDV has been demonstrated to be nonpathogenic inhumans, this virus is highly suited for use as a live vaccine.

In this regard, the use of genetically engineered NDV (vectors) forvaccine purposes may desire the presence of attenuation characteristicsin these strains. The introduction of appropriate mutations (e.g.,deletions) into the templates used for transfection may provide thenovel viruses with attenuation characteristics. For example, specificmissense mutations which are associated with temperature sensitivity orcold adaption can be made into deletion mutations. These mutationsshould be more stable than the point mutations associated with cold ortemperature sensitive mutants and reversion frequencies should beextremely low.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed. Such viruses would go through only one or a few rounds ofreplication within the host. When used as a vaccine, the recombinantvirus would go through limited replication cycle(s) and induce asufficient level of immune response but it would not go further in thehuman host and cause disease. Recombinant viruses lacking one or more ofthe NDV genes or possessing mutated NDV genes would not be able toundergo successive rounds of replication. Defective viruses can beproduced in cell lines which permanently express such a gene(s). Viruseslacking an essential gene(s) will be replicated in these cell lines butwhen administered to the human host will not be able to complete a roundof replication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse. Alternatively, larger quantities of the strains could beadministered, so that these preparations serve as inactivated (killed)virus vaccines. For inactivated vaccines, it is preferred that theheterologous gene product be expressed as a viral component, so that thegene product is associated with the virion. The advantage of suchpreparations is that they contain native proteins and do not undergoinactivation by treatment with formalin or other agents used in themanufacturing of killed virus vaccines. Alternatively, mutated NDV madefrom cDNA may be highly attenuated so that it replicates for only a fewrounds.

In another embodiment of this aspect of the invention, inactivatedvaccine formulations may be prepared using conventional techniques to“kill” the chimeric viruses. Inactivated vaccines are “dead” in thesense that their infectivity has been destroyed. Ideally, theinfectivity of the virus is destroyed without affecting itsimmunogenicity. In order to prepare inactivated vaccines, the chimericvirus may be grown in cell culture or in the allantois of the chickembryo, purified by zonal ultracentrifugation, inactivated byformaldehyde or β-propiolactone, and pooled. The resulting vaccine isusually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. It may be preferable to introduce the chimeric virusvaccine formulation via the natural route of infection of the pathogenfor which the vaccine is designed.

6. EXAMPLE Expression and Packaging of A Foreign Gene By Recombinant NDV

The expression of the chloramphenicol transferase gene (CAT) using theNDV minigenome is described. The NDV minigenome was prepared usingpNDVCAT, a recombinant plasmid containing the CAT gene. The pNDVCATplasmid is a pUC19 plasmid containing in sequence: the T7-promoter; the5′-end of the NDV genomic RNA comprising 191 nucleotides of noncodingNDV RNA sequence; 5 inserted nucleotides (3′CTTAA); the complete codingsequence of the chloramphenicol transferase (CAT) gene in the reversedand complemented order; the 3′- end of the NDV genomic RNA sequencecomprising 121 nucleotides of noncoding NDV RNA sequence; a BbsI cloningsite and several restriction sites allowing run-off transcription of thetemplate. The pNDVCAT can be transcribed using T7 polymerase to createan RNA with Newcastle disease viral-sense flanking sequences around aCAT gene in reversed orientation.

The length of a paramyxovirus RNA can be a major factor that determinesthe level of RNA replication, with genome replication being mostefficient when the total number of nucleotides is a multiple of six. ForNDV, the question of whether this rule of six is critical forreplication was examined by generating CAT mini-replicons of varyinglengths, differing by one to five nucleotides. Only one construct whosegenome was divisible by six was able to induce high CAT activity.

6.1. Construction of the Newcastle Disease Virus Minigenome

In order to construct an NDV minigenome, as described supra, thefollowing strategy was used. The 5′ terminal sequence of genomic NDV RNAwas obtained by RACE (Gibco, BRL) using standard techniques in the art.The template for the RACE reaction was genomic RNA which was purifiedfrom NDV virions (NDV-CL:California/11914/1944-like). As illustrated inFIG. 3, this terminal sequence comprised 64 nucleotides of a trailersequence plus 127 nucleotides of the untranslated region of the L gene.Located adjacent to the 191 viral nucleotide sequence, a 5 nucleotidesequence (3′CCTTAA) was inserted. A CAT gene comprised 667 nucleotidesof the CAT open reading frame which was placed between the viral 5′ and3′ terminal non-coding regions. In order to obtain the 3′ terminalregion of the NDV sequence, RT-PCR was used. The template for the RT-PCRreaction was in vitro polyadenylated genomic RNA of NDV. As illustratedin FIG. 3, the 3′ terminal region of 121 nucleotides was comprised of 56nucleotides of the untranslated region of the NP gene plus 65nucleotides of a leader sequence. The resulting construct of the NDVminigenome is shown in FIG. 1. Nucleotide sequences of 3′ and 5′non-coding terminal region shown in FIG. 3

6.2. Construction of the NDV NP, P & L Expression Plasmids

As described in Section 5, the transcription or replication of anegative strand RNA genome requires several protein components to bebrought in with the virus, including the L protein, P protein and NPprotein. In order to facilitate the expression from the NDV minigenome,the genes encoding each of the L, P and NP proteins were cloned intopTM1 expression vectors as illustrated in FIGS. 2A-C. The pTM1expression vectors comprises a T7 promoter, several cloning sites forinsertion of the gene of interest (L, P or NP), a T7 terminator, a pUC19origin of replication and an ampicillin resistance gene. In order toconstruct the expression plasmids, full length DNA of NDV nucleoprotein(NP), phosphoprotein (P) and polymerase (L) were obtained by RT-PCRamplification. These DNAs were cloned into T7 polymerase expressionvector pTM1, respectively (FIGS. 2A-C).

6.3. RNA Transcription of the NDV Minigenome

RNA transcription from the NDV minigene plasmid was performed with theRibomax kit (Promega) as specified by the manuscripts. In order to allowrun-off transcription, 1 μg of NDV minigenome plasmid (pNDVCAT) wasdigested with Bbs I. The linearized plasmid was then used as a templateof transcription reaction (for 2 hours at 37° C.). In order to removetemplate DNA, the resulting reaction mixture was treated with RNase-freeDNase (for 15 min. at 37° C.) and purified by phenol-chloroformextraction, followed by ethanol precipitation.

6.4. Cell Transfections

Cos-1 cells, or 293T cells were grown on 35 mm dishes and infected withthe helper virus rVV T7 at a multiplicity of infection (moi) ofapproximately 1 for 1 hour before transfection. The cells were thentransfected with the expression vectors encoding the NP, P and Lproteins of NDV. Specifically, transfections were performed with DOTAP(Boehringer Mannheim). Following helper virus infection, cells weretransfected with the pTM1-NP (1 μg), pTM1-P (1 μg) and pTM1-L (0.1 μg)for 4 hours. Control transfections, lacking the L protein, wereperformed on a parallel set of cells with pTM1-NP (1 μg), pTM1-P (1 μg)and mock pTM1-L (0 μg). After the 4 hour incubation period, cells weresubjected to RNA transfection with 0.5 μg of the NDV-CAT chimeric (−)RNA (see FIG. 1). Following RNA transfection, cells were allowed toincubate for 18 hours. The cell lysates were subsequently harvested forthe CAT assay.

6.5. Cat Assays

CAT assays were done according to standard procedures, adapted fromGorman et al., 1982, Mol. Cell. Biol. 2: 1044-1051. The assays contained10 μl of ¹⁴C chloramphenicol (0.5 μCi; 8.3 nM; NEN), 20 μl of 40 mMacetyl CoA (Boehringer) and 50 μl of cell extracts in 0.25 M Tris buffer(pH 7.5). Incubation times were 16-18 hours.

6.6. Results

In each cell line transfected with the NP, P, L expression vectors, andthe chimeric NDV-CAT RNA, high levels of expression of CAT was obtained18 hours post-infection. In addition, control transfected cells lackingthe L protein did not express CAT.

7. Rescue of Infectious NDV Viruses Using RNA Derived from SpecificRecombinant DNA

The experiments described in the subsections below demonstrate therescue of infectious NDV using RNA which is derived from specificrecombinant DNAs. RNAs corresponding to the chimeric NDV-CAT RNA may beused to show that the 191 nucleotides of the 5′ terminal and the 121nucleotides of the 3′ terminal nucleotides of the viral RNAs contain allthe signals necessary for transcription, replication and packaging ofmodel NDV RNAs. RNAs containing all the transcriptional units of the NDVgenomes can be expressed from transfected plasmids. Thus, thistechnology allows the engineering of infectious NDV viruses using cDNAclones and site-specific mutagenesis of their genomes. Furthermore, thistechnology may allow for the construction of infectious chimeric NDVviruses which can be used as efficient vectors for gene expression intissue culture, animals or man.

8. EXAMPLE Recombinant Newcastle Disease Virus Containing an HIV Antigengp160 Epitope inserted into the NDV Genome

In the Example presented herein, a chimeric NDV is constructed toexpress a heterologous antigen derived from gp160 of HIV. Theexperiments described in the subsections below demonstrate the use of arecombinant RNA template to generate a chimeric NDV that expresses a HIVgp160-derived peptide within the NDV genome and, further, this chimericNDV is used to elicit a vertebrate humoral and cell-mediated immuneresponse.

8.1. Construction of Plasmid

Recombinant NDV cDNA clones expressing HIV gp160 proteins may beconstructed in a number of ways known in the art. For example, asillustrated in FIG. 4, the HIV Env and Gag proteins may be inserted intothe NDV in a number of locations. In one example, the Env and Gagproteins are inserted between the M and L genes. In a different example,the Env and Gag proteins are inserted 3′ to the NP gene (between theleader sequence and NP). Alternatively, these HIV proteins will beincorporated between the NDV envelope proteins (HN and F) at the 3′ end.These proteins may also be inserted into or between any of the NDVgenes.

8.2. Generation of Infectious Chimeric Virus

Transfection of RNA derived from plasmid comprising a recombinant NDVgenome may be transfected into cells such as, for example, COS, 293 MDBKand selection of infectious chimeric virus may be done as previouslydescribed. See U.S. Pat. No. 5,166,057, incorporated herein by referencein its entirety. The resulting RNA may be transfected into cellsinfected with wild type virus by using standard transfection protocolprocedures. Posttransfection, the supernatant may be collected and usedat different dilutions to infect fresh cells in the presence of NDVantiserum. The supematant may also be used for plaque assays in thepresence of the same antiserum. The rescued virus can then be purifiedand characterized, and used, for example, in antibody production.

8.3. Hemagglutination Inhibition and Virus Neutralization Assays

Hemagglutination inhibition (HI) assays are performed as previouslydescribed (Palmer, D. F. et al., 1975, Immunol. Ser. 6:51-52).Monoclonal antibodies (2G9, 4B2, 2F1O, 25-5) are prepared by standardprocedures with a human anti-gp120 monoclonal antibody. Ascites fluidcontaining monoclonal antibodies is treated with receptor-destroyingenzyme as previously described (Palmer, D. F. et al., 1975, Immunol.Ser. 6:51-52).

For virus neutralization assay, cells in 30-mm-diameter dishes areinfected virus. After a 1 h adsorption, agar overlay containing antibodyat different dilutions is added. The cell monolayer is then stained with0.1% crystal violet at 72 h postinfection.

8.4. Immunization

6 weeks old BALB/c mice are infected either via the aerosol route withthe virus, or are immunized intraperitoneally (i.p.) with 10 μg ofpurified virus. For all booster immunizations, 10 μg of purified virusis administered i.p. Sera is collected 7 days after each immunization.

8.5. Radioimmunoassay

The radioimmunoassay is performed as previously described (Zaghouani, H.et al., 1991, Proc. Natl. Acad. Sci. USA 88:5645-6549). Briefly,microtiter plates are coated with 5 μg/ml peptide-BSA conjugate,saturated with 2% BSA in phosphate-buffered saline (PBS) and incubatedwith various dilution of serum. Bound antibodies are revealed by using¹²⁵I labelled antimouse kappa monoclonal antibody.

8.6. Radioimmunoprecipitation

The H9 human T cell line is acutely infected with HIV. Four dayspostinfection, 5×10⁷ infected cells are labelled with ³⁵S-cysteine,³⁵S-methionine, and ³H-isoleucine at 2×10⁶/ml in media containing 100μCi of each isotope per ml. After 20 h of metabolic labelling, theradioactive virions are pelleted by centrifugation for 1 h at 45,000rpm. The pellet is then resuspended in 1.0 ml of lysis buffer containing1% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride (PMSF).Approximately 20 μl of sera or 0.5 μg of monoclonal antibody (in 20 μlPBS) and 175 μl of virion lysate are incubated overnight at 4° C. in 0.5ml immunoprecipitation buffer containing 0.5% sodium dodecyl sulfate(SDS), 1 mg/ml BSA, 2% Triton X-100, and 50 mM sodium phosphate (pH7.4). The antigen-antibody complexes are bound to protein A-Sepharosebeads, and are analyzed by electrophoresis on a 10% SDS-polyacrylamidegel.

8.7. HIV-1 Neutralization Assays

The in vitro neutralization assay are performed as described previously(Nara, P. L. et al., 1987, AIDS Res. Hum. Retroviruses 3:283-302).Briefly, serial twofold dilutions of heat-inactivated serum areincubated for 1 h at room temperature with 150-200 syncytium formingunits of HIV virus produced in H9 cells. The virus/serum mixture isincubated for 1 h at 37° C. with 50,000 DEAE-dextran treated CEMss cells(adhered to microplate dishes using poly-L-lysine), or 50,000 H9suspension cells. After virus adsorption, the unbound virus is removedand 200 μl of media is added to each well. Four days postinfection, 50μl of supernatant media is removed for viral p24^(gag) proteinquantitation (Coulter Source, Inc.). The total number of syncytia inCEMss cells is counted five days postinfection. The neutralizationtiters are calculated by comparison with control wells of virus only,and are expressed as the reciprocal of the highest serum dilution whichreduced syncytia numbers by more than 50% or inhibited the p24 synthesisby more than 50%.

8.8. Induction of CTL Response

BALB/c mice is immunized with 0.2 ml viral suspension containing 10⁷ PFUof chimeric NDV virus. 7 days later, spleen cells are obtained andrestimulated in vitro for 5 days with irradiated spleen cells, alone orcoated with immunogenic peptides, in the presence of 10% concanavalin Ain the supernatant as previously described (Zaghouani, H. et al., 1992,J. Immunol. 148:3604-3609).

8.9. Cytolysis Assay

The target cells coated with peptides are labeled with Na⁵¹Cr₄ (100μCi/10⁶ cells) for 1 h at 37° C. After being washed twice, the cells aretransferred to V-bottom 96-well plates, the effector cells are added,and incubated at 37° C. in 7% CO₂. Four hours later, the supernatant isharvested and counted. The maximum chromium release is determined byincubating the cells with 1% Nonidet P40 detergent. The percentage ofspecific lysis is calculated according to the following formula: [(cpmsamples−cpm spontaneous release)/(cpm maximum release−cpm spontaneousrelease)]×100.

9. Intracellular Expression of Chimeric NDV-CAT RNA

In order to increase the efficiency of expression of NDV minigenomes, aplasmid (pT7-NDV-CAT-RB) was constructed for intracellular expression ofNDV-CAT RNA. This was achieved by inserting a ribozyme derived fromhepatitis delta virus directly after the end of the 3′ noncoding regionof the NDV-CAT RNA. Cotransfection of pTM1-NP, pTM1-P, pTM1-L andpT7-NDV-CAT-RT) into 293, 293T, COS1, CV1, or chicken embryo fibroblast(CEF) cells which were previously infected with rVV-T7 or with modifiedAnkara vaccinia virus expressing T7 polymerase (MVA-T7) resulted in highlevels of CAT activity (FIG. 6). CAT activity was approximately 100 to1,000 times higher than that achieved by direct RNA transfection of theNDV-CAT RNA.

10. Rescue of Infectious NDV Virus Using RNA Derived SpecificRecombinant DNA

In order to achieve rescue recombinant virus from a non-virus dependent,plasmid derived system, a plasmid allowing intracellular expression ofthe full-length antigenome of NDV was assembled. The NDV cDNA wasRT-PCRed in several pieces from purified RNA of a California-like strainof NDV (NDV-CL)(Meindl et al., 1974 Virology 58:457-463). The cDNApieces were ligated and assembled into a plasmid with T7 promoter andribozyme flanking sequences, resulting in plasmid pT7-NDV+RB. A silentmutation creating a new Xmal restriction site was introduced into the Lopen reading frame of pT7-NDV+-RB. CEF cell monolayers in 10 cm disheswere infected with MVA-T7 at a multiplicity of infection ofapproximately 0.1. One hour later, cells were transfected (lipofected)with 2.4 μg of pTM1-NP, 1.2 μg of pTM1-P, 1.2 μg of pTM1L and 1.5 μg ofpT7-NDV+-RB. After 8 h of incubation at 37° C., fresh medium was added.20 h postransfection, the vaccinia virus inhibitor araC was added at afinal concentration of 60 μg/ml. Two days postransfection, fresh mediumcontaining 100 μg/ml of araC was added. Supernatant from transfectedcells at a day 4 postransfection was used to inoculate the allantoicchamber of 10-days-old embryonated chicken eggs. After two days ofincubation at 37° C., the allantoic fluid was harvested and found to bepositive for the presence of NDV-CAT virus by hemagglutination. Analysisof the RNA isolated from the rescued virus confirmed the presence of thenewly inserted Xmal site, confirming that the virus was derived from thecloned plasmid cDNA. A schematic representation of the rescue procedureis protocol is shown in FIG. 7.

11. Expression of a Foreign Gene from an Internal Cistron of a ChimericNDV Genome

Plasmid pT7-NDV+-CAT/RN-RB was constructed by substituting the HN openreading frame in NDV-CL cDNA with the CAT open reading frame. Additionalextra nucleotides were added into the noncoding regions to allow for atotal nucleotide length of the resulting chimeric NDV RNA that wasdivisible by six. Cotransfection of pT7-NDV+CAT/HN-RB together withpTM1-NP, pTM1-P and pTM1-L into CEF monolayers that were previouslyinfected with MVA-T7 virus resulted in CAT activity as measured at day2postransfection (FIG. 8). These results demonstrate that it is possibleto use NDV as a vector for expression of foreign genes cloned astranscriptional units into the NDV genome.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any constructs, viruses orenzymes which are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference in theentirety for all purposes.

1. A recombinant RNA molecule comprising a binding site specific for anRNA polymerase of a Newcastle disease virus and signals required for NDVmediated replication and transcription, operatively linked to aheterologous RNA sequence, wherein the signals required for NDV mediatedreplication and transcription is contained in the 3′ and 5′ -noncodingflanking regions of the NDV viral RNA genome.
 2. A recombinant RNAmolecule comprising a binding site for an RNA polymerase of a Newcastledisease virus and signals required for NDV mediated replication andtranscription, operatively linked to a Newcastle disease viral gene;wherein the RNA molecule contains a mutation, wherein the signalsrequired for NDV mediated replication and transcription is contained inthe 3′ and 5′ -noncoding flanking regions of the NDV viral RNA genome.3. A recombinant cell comprising nucleotide sequences encoding arecombinant RNA molecule Of claim 1 or 2 and NDV RNA polymersaseproteins P and L.
 4. The recombinant RNA molecule of claim 1 or 2 inwhich the polymerase binding site comprises the polymerase binding sitecontained in the 3′ and 5′ -noncoding flanking region of a Newcastledisease viral RNA genome.
 5. The recombinant RNA molecule of claim 1 or2 in which the 3′ and 5′ -noncoding flanking regions have a genomic NDVviral sense sequence as shown in SEQ ID NO: 2 and SEQ ID NO: 1,respectively.
 6. The recombinant RNA molecule of claim 1 in which theheterologous RNA encodes a viral antigen.
 7. The recombinant RNAmolecule of claim 6 in which the viral antigen is derived from humanimmunodeficiency virus, Newcastle disease virus, influenza, respiratorysyncytial virus, Marek's disease virus, infectious bursal disease virus,infectious bronchitis virus, infectious bursitis virus, chicken anemiavirus, infectious laryngotracheitis virus, avian leukosis virus,reticuloendotheliosis virus, avian influenza virus, rabies virus, felinedistemper virus, vesicular stomatitis virus, rinderpest virus, orswinepox virus.
 8. A method for producing a chimeric negative-strand RNAvirus, comprising transfecting a host cell with nucleotide sequencesencoding the recombinant RNA of claim 1 or 2 and the viral functionsrequired for replication and transcription, and recovering the chimericvirus from the culture.